Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Agar and ammonium nitrate influence hyperhydricity,tissue nitrate and total nitrogen content of serviceberry (Amelanchier arborea) shoots in vitro

Shoot tip cultures of Amelanchier arborea Michx.f. were grown on Murashige & Skoog or Woody Plant (WP) medium containing 4.4 M benzyladenine and various concentrations of agar. Increases in agar concentration affected various culture growth variables, decreased culture hyperhydricity and increased tissue nitrate concentration. Additions of ammonium nitrate to cultures grown on WP medium containing 0.4% agar increased all growth variables measured except percent dry weight. Hyperhydricity and tissue nitrate concentration also increase in response to increasing ammonium nitrate in the medium. Since hyperhydricity was shown to be both positively and negatively correlated with increases in tissue nitrate content, it is unlikely that tissue nitrate level alone directly affects hyperhydricity.Abbreviations BA benzyladenine - MS Murashige & Skoog - WP Woody Plant     

The use of thidiazuron in tissue culture

Summary Thidiazuron (N-phenyl-N’-1,2,3-thiadiazol-5-ylurea) was first reported to have cytokinin activity in 1982. Since then, thidiazuron has been used successfully in vitro to induce adventitious shoot formation and to promote axillary shoot proliferation. Thidiazuron is especially effective with recalcitrant woody species. Shoot numbers produced on medium containing thidiazuron are equivalent to or greater than numbers initiated on medium with purine-type cytokinins. Low concentrations of thidiazuron (0.0022 to 0.088 mg/liter) are effective for micropropagation. Prolonged exposure to thidiazuron should be avoided, as this may cause hyperhydricity, abnormal shoot morphology, or problems in rooting. Presented in the Session-in-Depth Novel Plant Growth Regulators at the 1992 World Congress on Cell and Tissue Culture, Washington, D.C., June 20–25, 1992.     

Minimizing growth regulators in shoot culture of an endangered plant,Hackelia venusta (Boraginaceae)

Summary Hackelia venusta (Boraginaceae) is an endangered perennial herb endemic to the interior northwestern United States. Because of seed scarcity, micropropagation (anex situ conservation strategy) could produce true-to-type plantlets suitable for reintroduction. We hypothesized that clones of predetermined size could be rapidly produced by supplementing multiplication and rooting media with minimal levels of cytokinin and auxin. Microshoots derived from shoot expants were cultured on Murashige and Skoog (1962) media supplemented with 1% (wt/vol) agar and 0.0001 to 10 μM benzyladenine. Inverse regression estimates on 3 genotypes predicted that a target of 2.5 axillary microshoots per explant would require a minimal level of 0.04±0.02 μM benzyladenine. Culture of 25 genotypes with 0.04 μM benzyladenine resulted in an average of 2.3±0.1 axillary microshoots per explant. Elongated microshoots were transferred to media supplemented with 0.1 to 25 μM indoleacetic acid. Clones rooted from 36% to 100% success after 4 wk in 2.0 μM indoleacetic acid. Plantlets transplantedex vitro with three or more roots survived at 84% versus 46% of plantlets with fewer roots. Up to 84% of the plantlets survived in a planting trial. The data suggest that shoot culture ofHackelia venusta, with minimal growth regulators, can produce axillary microshoots for reintroduction.     

Matric potential evaluations and measurements for gelled substrates

A new method for evaluating the matric potential of gelled media has been developed. The method allows the derivation of the matric potential as a limit of a series of measurements of water potential values from gelled media prepared without added components, from agar powders progressively cleaned of mineral impurities. Three commercial agar brands were tested, and for these the matric potential was found to contribute only between 1 and 2% of their total water potential. Thermodynamic features relating matric and osmotic potentials are described. New hypotheses for understanding the water flux mechanism from gel to tissue cultured explants are discussed. Movement of water along polymeric chains is postulated to be a facilitated step in comparison with bulk movement.     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743